There are few techniques that can both rapidly and reversibly manipulate components inside living cells;furthermore, the development of tools capable of doing so will significantly enhance the ability of scientists to understand biology and treat disease. The long-term goal of this research is to engineer new molecular tools that can be assembled and operated inside biological environments such as the cell. Towards this goal, this proposal describes an investigation of the phase transition of environmentally-responsive polypeptides (ERPs). The hypothesis is that ERPs can sequester key factors inside cells and that they can reversibly switch on and off molecular pathways inside live cells. To demonstrate the feasibility of this approach, ERPs will be designed to control a ubiquitous cellular process called clathrin-mediated endocytosis. This process is important in the regulation of many diseases, including cancer and infection. The following specific aims are proposed: 1) Biosynthesis and biophysical chemistry of ERP switches: A biophysical approach will be used to characterize the behavior of ERPs and ERP-fusion in solution and mathematical modeling will be developed that permits these systems to be designed to response to any desired temperature. The intracellular behavior of ERPs will be observed inside human cells that produce ERPs fused to a fluorescent protein that can be viewed under a microscope. Using this construct, we will evaluate how quickly the ERPs self-associate inside the cell. A phase diagram describing the parameters that influence the intracellular ERP phase transition will be compared to that in free solution. 2) ERP switching of clathrin-mediated endocytosis: ERPs will be fused to a key protein involved with the uptake of factors in the cellular environment. This research project is intended to culminate in a general strategy for selectively halting cellular pathways. This proposal is innovative in three main ways: (i) the specific ERP behavior proposed here has never been observed inside of live human cells;(ii) this approach is expected to be a rapid, reversible technique that can potentially switch on or off specific cell pathways;and (iii) this approach can be generalized to target any cellular pathway for which a known protein interacts. The successful demonstration of this approach is intended to shift the paradigm for how cellular biology studies are performed, enabling precise manipulation of biological processes that are fundamentally important to the treatment of disease. PUBLIC HEALTH RELEVANCE: Understanding the process by which diseases, such as cancer or infection, proceed at a cellular level is critical to the development of new treatments. This proposal describes the exploration of a novel, enabling technology that is intended to rapidly turn on and off critical cellular process involved with disease. Success of this project will catalyze numerous future studies of many disease processes and culminate with improved treatments for illnesses.